This proposal examines the role of the non-receptor tyrosine kinase Src and related Src family kinase (SFKs) during vascular responses induced by vascular endothelial growth/permeability factor (VEGF/VPF). Recently, a requirement has been identified for specific SFKs during VEGF-induced vascular permeability (VP) in vivo. VEGF-induced VP is physiologically relevant in hypoxic tissues, tumor growth, and is associated with the extravasation of plasma proteins from the circulation and in tumor metastasis. Preliminary studies suggest that Src is essential for integrin alpha-v-beta5-dependent VEGF-induced vascular responses such as VP. The mechanism of action appears to involve the formation of a complex between phosphorylated FAK and alpha-v-beta5, which in this case, is dependent on Src kinase activity. However, SFKs are multifunctional proteins, consisting of several distinct domains (a single unique, SH3, SH2 and kinase domains). The function of these domains in VEGF-induced vascular responses, other than cell proliferation and migration in general, are poorly understood. This proposal examines the domain requirement for the selective Src-requirement for the VEGF-induced VP vascular response in vivo and in vitro. The characterization of VEGF responses in VP-defective animals (i.e. src-/- mice) will, for the first time, enable the distinction between the capacity of VEGF to function as a growth factor to promote migration and proliferation from its function to promote VP of the vascular endothelium. In Aim 1 of this proposal, the molecular basis of the SFK requirement for VEGF-induced VP will be examined in primary human endothelial cells. The requirement for specific Src domains in VEGF-induced VP, migration and proliferation will be compared with the capacity for endothelial cells isolated from src-/- or control mice to support VEGF-induced vascular responses. In Aim 2, src -/- and control mice will be used as hosts for syngeneic and transgenic tumor growth studies. These experiments will test the hypothesis that VP defects may influence the deposition of extracellular matrix components from the circulation into the tumor microenvironment, thereby reducing tumor metastasis in VP-defective mice. These studies will provide the basis to determine in Aim 3 which SFK domain are sufficient to restore VP defects in src-/- mice using a transgenic germline rescue strategy. In combination, these studies are designed to provide an insight into the molecular mechanism of Src-mediated VP following VEGF stimulation of blood vessels and during tumor growth and metastasis.